The contribution to the free energy of binding
of each of the residues forming the binding site for a
human IgG Fc fragment on the surface of the B1 domain of
protein G was determined by alanine-scanning mutagenesis.
The interface between these two proteins is atypical in
that it is smaller than usual, polar in character, and
involves two well-defined “knobs-into-holes”
interactions. The bulk of the free energy of binding is
contributed by three central residues, which make hydrogen
bonds across the interface. Of these, the most critical
interaction is formed by Glu27, which acts as a charged
knob on the surface of the B1 domain, inserting into a
polar hole on the Fc fragment. A single alanine mutation
of this residue virtually abolishes stable complex formation.
Formation of a stable interface between these two proteins
is therefore dominated by a small, polar “hot spot.”